1. Field of the Invention
The present invention is related to a method for detecting a target biomolecule in a test sample by adding an internal control biomolecule to the test sample; to a negative control sample, to a positive control sample and to a reagent control sample or adding an internal control biomolecule to the test sample, to a negative control sample, to a positive control sample comprising the target biomolecule and providing a reagent control sample comprising the target biomolecule, determining in each sample a signal, and verifying the signal thereby detecting the target biomolecule. The invention is also related to a method for verifying the determination of a signal indicating the presence of a target biomolecule. The invention is further related to a method for detecting the presence or the absence of a member of a group of target nucleic acids in a sample and a method for verifying the determination of a signal indicating the presence of a member of a group of target nucleic acids. Uses and kits are considered as well.
2. Description of Related Art
The determination of nucleic acids has become an important tool in analytical chemistry, especially in health care. For example, infection diseases and genetic status can be easily determined on the basis of the presence or the amount of a nucleic acid indicative of said disease or status in samples received from the individual. For this reason methods were established using sequence specific hybridization of a nucleic acid, preferably an oligonucleotide, with a target nucleic acid indicative for that disease or genetic status. Many target nucleic acids are present in an organism in such low concentration, that a direct detection in a sample derived from that organism is not possible. Such targets need to be amplified before detection. Suitable amplification methods are for example LCR (U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308; WO 90/01069; WO 89/12696; and WO 89/09835), cycling probe technology (U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/14106, and WO 95/00667), Invader™ technology (U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669), Q-Beta replicase technology (U.S. Pat. No. 4,786,600), NASBA (U.S. Pat. No. 5,409,818; EP-0 329 822), TMA (U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365, 5,710,029), SDA (U.S. Pat. Nos. 5,455,166 and 5,130,238) and PCR (U.S. Pat. No. 4,683,202).
In order to minimize false results in nucleic acid determinations, authorities in several countries require the use of control nucleic acids. Especially when using amplification methods such control nucleic acids are very important, because the amplification process can be strongly influenced by the reaction conditions, which could lead to misleading results. Sometimes inhibitory substances are contained in a sample, which could lead to false negative results. A review of control concepts is provided by Valentine-Thon, E., J. Clinical Virol. 25 (2002) S13-S21.
In general one can distinguish external and internal controls. External controls, like classical positive and negative controls, mimic positive and negative samples and are normally used to check whether the assay runs properly or whether contaminants are contained. An internal control for example is useful for recognizing inhibitory substances possibly contained in a sample or can be used as a quantification standard in a quantitative assay. In contrast to an external control, which normally is tested in a separate reaction chamber, an internal control is preferably incubated in the same reaction chamber together with the analyte to be tested. Therefore, the control or the amplified product of that control has to be distinguishable from the analyte or from the amplified product of that analyte. When using an amplification method an internal control nucleic acid is being co-amplified essentially under the same reaction conditions as the target nucleic acid. These conditions include reagent concentrations, temperature, inhibitor concentration or enzymatic activities. Frequently used sequences for controls are derived from housekeeping genes (see Chelly, J., et al., Eur. J. Biochem. 187 (1990) 691-698; Mallet, F., et al., J. Clin. Microbiol. 33 (1995) 3201-3208), but also non-natural sequences are being used (see e.g. EP 1 236 805).
The amplified nucleic acid derived from the internal control can be distinguished from the amplified nucleic acid derived from the target nucleic acid for example by their different length or hybridization capability to a distinct probe (for reviews see: Clementi, M., et al., PCR Methods Applic. 2 (1993) 191-196; Clementi, M., et al., Arch. Virol. 140 (1995) 1523-1539). In all cases the nucleotide sequence of the internal control is partially or totally different from the target nucleic acid sequence (see also Haentjens-Herwegh, S., et al., Recent Res. Devel. Microbiology 4 (2000) 547-556).
One of the most critical aspects in an amplification reaction is the binding of the primer to the target nucleic acid. Therefore internal controls are being used, which have the same primer binding sites as the target nucleic acid (see for example Gilliland, G., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 2725-2729).
WO 02/18635 and WO 99/06594 disclose the use of encapsulated internal control sequences mimicking nucleic acids encapsulated in a virus shell.
EP 1 319 716 discloses the use of several discriminable internal control nucleic acids for testing nucleic acid isolation and amplification.
WO 2004/055205 discloses the addition of internal control sequences to a sample which undergoes sample preparation. Then the signal from the IC is compared to external controls which did not undergo sample preparation in order to verify the efficiency of cell lysis and of sample preparation as well as the performance of nucleic acid amplification and/or detection.
U.S. Pat. No. 6,277,560 discloses the use of the DNA of an external microorganism as external standard for quantification and the use of an internal control for evaluating the efficiency of nucleic acid amplification.
Gilliland, G., et al., Proc. Natl. Acad. Sci. USA 87 (1990) 2725-2729 discloses the use of negative controls and internal controls for quantification.
Brakenhoff, R., et al., Clin. Cancer Res. 5 (1999) 725-732 disclose a PCR assay with an internal standard for RNA quality control, an external standard for sensitivity control and a negative control. WO 2005/061737 discloses a kit with an internal control, a positive control and a negative control.
Leushner and Kelly, Qiagennews, No. 4, 2000, page 21, disclose a method for detection of pathogenic enteric bacteris in stool by multiplex PCR using an internal control and a positive control.
The “LightCycler® foodproof E. coli 0157 Detection Kit” manufactured by Roche Applied Sciences (Mannheim, Germany) discloses in its manual a PCR method for the qualitative detection of E. coli serotype 0157 using an internal control and a control template.
US2003/077622 relates to methods and compositions that provide a positive control to identify inhibition during a signal amplification reaction.
The “CT/NG Test for Chlamydia trachomatis” manufactured by Roche Molecular Systems (Branchburg, N.J., USA) discloses in its manual a PCR method for detection of Chlamydia trachomatis using an internal control and the C. trachomatis target DNA.
The manual for the CycleavePCR™ meat species identification kit manufactured by TaKaRa, Japan, discloses a method for the differentiation of different meat species including a control concept.